Ethylnitrosourea (ENU) is a powerful transplacental carcinogen. A single dose to a pregnant rat produces neural tumors in adult offspring many months later. These tumors, routinely produced in our department, have been propagated in cell culture. In cell lines from ENU tumors we have compared chromosome constitution, growth characteristics in culture, histological findings of the tumor and tumorigenicity of the cells. In all 12 ENU-induced neural tumor lines (schwannomas and gliomas), excess chromosome 4 was present as a common chromosome finding along with non-specific changes, indicating that extra chromosome 4 material can be used as a marker for ENU neural tumors. It has also been demonstrated that tumorigenic doses of ENU produce chromosome aberrations in bone marrow and fetal tissues of treated animals as well as in cultured cell in vitro. Although the chromosome breaking effects disappear they suggest that a mutation could be an indication of and possibly an explanation for the oncogenic effect. Currently it is of interest to determine whether excess chromosome 4 is related to ENU acting as a specific etiologic agent by studying the chromosome constitution of non-neural ENU tumors, or whether the chromosome is associated with a particular tumor type by studying rat brain tumors induced by other agents. It is also important to demonstrate the excess 4 in direct preparations of tumors without cell culture. Our main objective, however, is to study early stages of oncogenesis in brain which is a prime target for eventual tumor. Brain will be removed from newborn rats treated with ENU transplacentally, and initiated in cell culture. At various stages, cells will be examined for oncogenic transformation, cytogenetic changes and injected into newborn hosts to test for tumorigenicity.